The goal of this work is identification of a biochemical function of calretinin (CR). The results obtained thus far describe come of the basic characteristics of CR as a calcium binding protein. These include the following: (1) CR undergoes a calcium-dependent conformational change. This was shown in two ways. First, changes in the intrinsic fluorescence (due to Trp) of CR and in the fluorescence of a probe attached to Cys residues on the protein were observed as a result tf Ca2+binding to CR. Secondly, different tryptic cleavage fragments of CR were obtained in the presence and absence of calcium as shown by identification of fragments using a combination of mass spectrometry and amino acid analyses. The mass spectroscopic analyses also revealed that CR is blocked at the N-terminus by a relatively small functional group. Examination of the behavior of the tryptic fragments by column chromatography revealed a Ca2+-sensitive hydrophobic region which might represent a site of interaction with target(s). The fragments were also used to identify one high affinity antibody recognition site; (2) Subcellular fractionation of rat cerebellum and examination of samples on immunoblots revealed separate patterns of localization for the three calcium binding proteins: CR, calbindin D28k and parvalbumin. Both calbindin D28k and CR were associated, to a smaller degree, with synaptic and microsomal membranes with partial calcium dependence of this binding, while parvalbumin was only detected in the cytosolic fraction. Calbindin D28k was enriched in the microsomal fraction of cerebellum but not in caudate; (3) Polyclonal and monoclonal calcium binding protein antisera may preferentially recognize antigens in a particular calcium-induced conformational state. Polyclonal antibodies specific to CR and a calbindin D28k monoclonal antisera recognized a Ca2+-free form (fixed with crosslinker) to a greater extent than the Ca2+-bound form (also fixed).